Glutathione and transition-metal homeostasis in Escherichia coli

Glutathione (GSH) and its derivative phytochelatin are important binding factors in transition-metal homeostasis in many eukaryotes. Here, we demonstrate that GSH is also involved in chromate, Zn(II), Cd(II), and Cu(II) homeostasis and resistance in Escherichia coli. While the loss of the ability to synthesize GSH influenced metal tolerance in wild-type cells only slightly, GSH was important for residual metal resistance in cells without metal efflux systems. In mutant cells without the P-type ATPase ZntA, the additional deletion of the GSH biosynthesis system led to a strong decrease in resistance to Cd(II) and Zn(II). Likewise, in mutant cells without the P-type ATPase CopA, the removal of GSH led to a strong decrease of Cu(II) resistance. The precursor of GSH, gamma-glutamylcysteine (gammaEC), was not able to compensate for a lack of GSH. On the contrary, gammaEC-containing cells were less copper and cadmium tolerant than cells that contained neither gammaEC nor GSH. Thus, GSH may play an important role in trace-element metabolism not only in higher organisms but also in bacteria.     

Rice PCR1 influences grain weight and Zn accumulation in grains

Proteins containing a placenta‐specific 8 domain (PLAC8) function as major organ size regulators in Solanum lycopersicum and Zea may, and putative metal ion transporters in Arabidopsis thaliana, Oryza sativa and Brassica juncea. However, it is unknown how PLAC8 domain‐containing proteins fulfill such diverse roles. Here, we found that plant cadmium resistance 1 (PCR1) influences both zinc (Zn) accumulation and grain weight in rice. OsPCR1 knockout and knockdown lines produced lighter grains than the wild type, while OsPCR1 overexpression lines produced heavier grains. Furthermore, the grains of OsPCR1 knockdown lines exhibited substantially higher Zn and lower cadmium (Cd) concentrations than the control, as did yeast heterologously expressing OsPCR1. Through sequence analysis, we showed that the amino acid sequence of japonica‐type PCR1 was distinct from that of indica‐type and wild rice accessions. This difference was correlated with distinct Zn‐related phenotypes. Japonica‐type PCR1 had a shorter N‐terminus than did PCR1 in the other rice types, and yeast heterologously expressing japonica‐type PCR1 was more sensitive to Zn than was yeast expressing indica‐type PCR1. Furthermore, japonica‐type grains accumulated less Zn than did indica‐type grains. Our study suggests that rice PCR1 maintains metal ion homeostasis and grain weight and might have been selected for during domestication.     

Functional characterisation of two phytochelatin synthases in rice (Oryza sativa cv. Milyang 117) that respond to cadmium stress

• Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised.
• To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl₂ using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15).
• The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana.
• These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

     

Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis

Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)‐chelating peptides and by sequestering PC–metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As–PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC₂–Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC₂–Cd transport activity compared with PC₂–As. In contrast, barley vacuoles readily accumulated comparable levels of PC₂–Cd and PC₂–As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC‐type transporters. Interestingly, barley vacuoles exhibited enhanced PC₂ transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non‐essential toxic metal(loid)s.     

Functional analyses of TaHMA2, a P1B‐type ATPase in wheat

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2‐transformed yeast. The over‐expression of TaHMA2 increased the elongation and decreased the seed‐setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over‐expression also improved root‐shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over‐expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over‐expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Molecular characterization of a rice metal tolerance protein, OsMTP1

Rice (Oryza sativa L. 'Nipponbare') cDNA subtractive suppression hybridization (SSH) libraries constructed using cadmium (Cd)-treated seedling roots were screened to isolate Cd-responsive genes. A cDNA clone, encoding the rice homolog of Metal Tolerance Protein (OsMTP1), was induced by Cd treatment. Plant MTPs belong to cation diffusion facilitator (CDF) protein family, which are widespread in bacteria, fungi, plants, and animals. OsMTP1 heterologous expression in yeast mutants showed that OsMTP1 was able to complement the mutant strains' hypersensitivity to Ni, Cd, and Zn, but not other metals including Co and Mn. OsMTP1 expression increased tolerance to Zn, Cd, and Ni in wild-type yeast BY4741 during the exponential growth phase. OsMTP1 fused to green fluorescent protein was localized in onion epidermal cell plasma membranes, consistent with an OsMTP1 function in heavy metal transporting. OsMTP1 dsRNAi mediated by transgenic assay in rice seedlings resulted in heavy metal sensitivity and changed the heavy metal accumulation in different organs of mature rice under low-concentration heavy metal stress. Taken together, our results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant.     

Transcriptome-wide identification and expression analyses of ABC transporters in dwarf polish wheat under metal stresses

ABC transporters, which comprise one of the largest protein families, are involved in maintaining osmotic homeostasis, nutrient uptake, pathogen resistance, and metal tolerance. In this study, 30 ABC genes in dwarf polish wheat were characterized and classified into seven subfamilies (ABCA - ABCG). Among them, 24 ABC transporters were newly found in wheat. The expressions of 13 ABC genes in roots and leaves under six metal stresses were also analyzed. All these genes were differentially regulated by Cd (except ABCE2, ABCF4, and ABCF6 in roots), suggesting that these genes participate in Cd transport, sequestration, or uptake. These genes were also differentially regulated by other metals including Cu, Mg, Zn, Fe, and Ni. Results suggest that the expressions of ABC transporters in dwarf polish wheat played important roles in metal transport and detoxification.     

The <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> NRAMP Homologue TcNRAMP3 is Capable of Divalent Cation Transport

The NRAMP gene family encodes integral membrane protein and mediates the transport of Fe, however, its function in transport of toxic metal ions is not very clear in plants. TcNRAMP3 was isolated from Thlaspi caerulescens, and encoded a metal transporter member of the NRAMP family. TcNRAMP3 was predominantly expressed in roots of T. caerulescens by semi-quantitative RT-PCR. The expression of TcNRAMP3 was induced by iron starvation and by the heavy metals Cd and Ni in roots. TcNRAMP3 was able to rescue growth of an iron uptake fet3fet4 mutant yeast strain, suggesting a possible role in iron transport. Expression of TcNRAMP3 in yeast increased Cd sensitivity and Cd content, while it enhanced the Ni resistance and reduced Ni accumulation, indicating that TcNRAMP3 could accumulate Cd and exclude Ni in yeast. Furthermore, overexpression of TcNRAMP3 in tobacco resulted in slight Cd sensitivity of root growth and did not influence Ni resistance. These results suggested that TcNRAMP3 played a role in metal cation homeostasis in plant.     

Isolation and Characterization of Four Gram-Positive Nickel-Tolerant Microorganisms from Contaminated Sediments

Microbial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared with pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes.     

From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro‐organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well‐known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium‐binding metallothioneins (CdMTs), higher heavy metal‐MT stoichiometry values, and a strictly conserved modular–submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole‐cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.